Reconstituted human oral and esophageal mucosa in culture

Abstract

We have successfully established monolayer and organotypic culture techniques for growing human oral and esophageal epithelial cells. Cells in monolayer culture were grown in serum-free medium, modified from techniques previously reported by our group. The organotypic cultures were grown in a defined medium supplemented with 10% fetal calf serum. Oral and esophageal cells were maintained in keratinocyte basal medium with pituitary extract and other supplements, and 0.05 mM calcium for 7-9 and 9-11 passages, respectively. Both cell types had similar morphology by phase contrast microscopy. When confluent, the cells were predominantly small, basaloid, and uniform and interspersed with larger, differentiated cells. By immunohistochemistry, both cell types in monolayer were positive to AE1, AE3, and 34BE12 antibodies to keratins of stratified epithelia. Oral epithelial cells in monolayer also were positive to 35BH11, representative of simple epithelial keratins, while esophageal cells were not. The esophageal cells were locally positive to K13, while the oral cells were negative. Both were negative for K19. When comparing monolayer to organotypic cultures and to in vivo specimens, there was a significant difference in the expression of keratins. Using organotypic cultures, AE1, AE3, and 34BE12 were strongly positive in both oral and esophageal cells, similar to in vivo tissues. In contrast to monolayers, both were also focally positive for K19. Esophageal cells were strongly positive for K13, while the oral cells were mildly but uniformly positive. Both were negative for keratins of simple epithelia. These two cell culture techniques offer unique opportunities to study the pathobiology, including carcinogenesis, of stable cell systems from the oral and esophageal epithelia.